Our long term goal is to understand how gene expression in higher cells is regulated at the level of chromatin structure, and how this "epigenetic" information is passed on to daughter cells. It may be crucial to know the details of chromatin structure and assembly to understand some aspects of cell biology at the molecular level. One aspect is cell malignancy. There is increasing evidence that some normal cells contain genes that are capable of inducing cell malignancy if these genes are expressed at abnormal levels or at the wrong time. Tissue-specific differences in chromatin structure are of primary interest because of the correlation with functional differences. One of the most apparent tissue-specific features of chromatin is the average nucleosome spacing periodicity (repeat length). The factors that determine this fundamental property of chromatin will be determined here using an in vitro chromatin assembly system. Such experiments were previously not possible. Quantitative information will be obtained from analyses of the periodicities of polyd(A-T) fragments. To study chromatin assembly on particular DNA regions or specific genes, two novel in vitro methods will be explored. One of these methods might permit the engineering of specific types of chromatin "constructs" that would be useful for functional studies. Additionally, DNA regions (of plasmids or cloned fragments) that can influence the positioning of nucleosome arrays will be identified and studied, using direct end-labeling and in vitro reconstitution methods. A possible relationship, which could be of functional interest, between "nucleosome-positioning sequences" and the integral nature of the nucleosome repeat will also be investigated.